Compositions for combating tuberculosis



Patented May 6, 1952 COMPOSITIONS FOR COMBATING TUBERCULOSIS Herman.Herbert Fox, Passaic, N. J., assignor to Hoffmann-La Roche Inc., RochePark, Nutley, N. J., a corporation of New Jersey No Drawing. ApplicationMarch 7, 1952, Serial No. 275,249

Claims. 1

My invention relates to compositions useful to N combat tuberculosis.More particularly, my invention relates to such compositions in whichthe active ingredient is isonicotinic acid hydrazide or an acid additionsalt thereof, such as the hydrochloride. This application is acontinuation-inpart of my co-pending application Serial No. 216,255,filed March 17, 1951.

It is known that tuberculosis is a contagious disease which usually runsa protracted course, often lasting for years before death or recovery.Relapses are common. It is one of the most important causes of prolongeddisability and chronic ill health. It is caused by the tubercle bacillus(Mycobaterium tuberculosis). Human tuberculosis is usually caused eitherby the human strain or the bovine (cattle) strain of the bacillus. Thebacillus is comparatively resistant. That is why dried sputum particlesretain their infectiousness for prolonged periods, thus facilitating thespread of the disease. Tuberculosis is usually acquired either by theinhalation of droplets of infected sputum, either fresh or dried, or byconsumption of milk from tuberculous cows.

Until the discovery of streptomycin, it was impossible to attack thetubercle bacillus directly.

pounds had previously been recommended, but

they were shown not to affect human tuberculosis. Streptomycin was thefirst drugv that could inhibit the bacillus in both animal and humanbodies. It is believed that streptomycin acts by inhibitingmultiplication of the bacillus. In this way it helps the recuperativepowers of the body to dispose of the disease. Streptomycin, however, hasa number of significant shortcomings: (1) it is not always welltolerated; (2) it may cause temporary or permanent deafness; (3) itcauses dizziness and vertigo; (4) it has to be given by injectionfand(5) the bacillus may learn to live with the drug, 1. e., it may becomeresistant to its effect so that it loses its therapeutic value. Patientsin which the therapeutic value of streptomycin is lost may stillcontinue to raise sputum containing the bacilli and these bacilli whichare resistant to streptomycin may affect other individuals with thedisease. The patients so infected Will'not respond because these germsare resistant to the drug.

Para-'amino-salicylic acid has been tried and has been found tobe a weakanti-tubercular agent. However, when used with streptomycin itreinforces the action of streptomycin and delays the appearance ofresistant strains of the bacillus. Para-amino-salicylic acid may causesevere and prolonged diarrhea. Patients may become resistant to thecombination. Moreover, there have been serious side reactions.

Nicotinamide and derivatives thereof have also been tried. The amide hasbeen reported to function only as a vitamin, and its derivatives werefound to have less activity than the amide. Moreover, hi h toxicity wasobserved. (J. Org. Chem, 13, 1948, Kushner et al., pp. 834-836; J. Lab.8: Clin. Med., 33, 1948, McKenzie et a1.,pp. 1249- 1253; Mfg. Chemist,20, 1949, p. 185.)

Among the objects of the present invention is the production ofcompositions for combatting tuberculosis in humans, poultry, cattle (e.g., cows), monkeys, and the like. My new compositiveness has been foundfor a variety of animals,

including poultry, cows, rabbits, cats, dogs, mice,

rats, guinea pigs, and the like. Significant results in the treatment ofhuman beings hav been noted, as will be brought out hereinafter.

One object of my invention is to provide isonicotinic acid hydrazidecompositions for use in com-batting tuberculosis. According to myinvention, this hydrazide may be associated with a carrier which may beeither a solid mate-rial or a sterile parenteral liquid. Thecompositions may take the form of tablets, powders, capsules, or otherdosage forms which are particularly useful a for oral ingestion. Liquiddiluents are employed in sterile condition for parenteral use, that is,by injection. Such a medium may be a sterile solvent such as water. theform of active material, namely, hydrazide material, admixed with soliddiluents and/or tableting adjuvants such as cornstarch, lactose, talc,stearic acid, magnesium stearate, gums, or the like. Any of thetableting materials used in pharmaceutical practice may be employedwhere there is no incompatibility with the isonicotinic acid hydrazidematerial. The material may be tableted with or without adjuvants.Alternatively the hydrazide material with its adjuvant material may beplaced in the usual capsule or resorbable material such as the usualgelatin capsule and administered in that form. In yetanother embodiment,the hydrazide composition may be put up into powder packets andemployed.

Or the hydrazide composition may be prepared in the form of a suspensionin a material in which the hydrazide is not soluble.

The compositions may take My invention embraces the use of isonicotinicacid hydrazide in various forms, e. g., as such, or in the form of acidaddition salts of which the hydrochloride is illustrative. The termhydrazide,

known methods. Meyer and Mally, Monatsh., 33, i

1912, page 400. Insofar as is known, however, its physiologicalproperties have not heretofore been investigated; nor has the compoundbeen applied for therapeutic purposes.

The hydrazide compound has been found to be highly active againsttuberculosis, in vivo as well as in vitro. In tuberculous infection ofmice, it exerts an antituberculous activity both against hematogenousinfection, that is, infection produced through the intravenous route, aswell as against the generally therapeutically more resistantbronchogenic infection, that is, infection produced through theintranasal route with M. tuberculosis. Thus, it has been found that byfeeding mice, hematogenously infected with M. tuberculosis, a dietcontaining as little as 1 gram ofisonicotinic acid hydrazide per 60kilograms of diet, or 0.0017 per cent, development of tuberculosislesions in the animals is prevented. In the caseofmice infected with M.tuberculosis by bronchogenic dissemination, as little as 0.0025 per centof isonicotinic acid hydrazide has been observed to prevent spreading oftuberculosis. Not only is the compound effective on administration bythe oral route, butit is also effective when administered parenterally,e. g., subcutaneously or intraperitoneally. The tolerated dose orally inmice is 125 mg. per kg. of body weight in a single dose andsubcutaneously 200 mg. per kg. in a single dose. In in vitro tests, itinhibited the growth of tubercle bacilli in concentrations of werecarried out on white mice weighing from 18-20 grams. A single strain ofmice'bred under uniform conditions of nutrition and environment was usedin all the experiments.

Example 1 One gram of isonicotinic acid hydrazide was ground to a finepowder and was then mixed with 25 grams of a commercially availablemouse diet known as the Rockland Mouse Diet, which is sold by ArcadyFarms Milling Company, Chicago, Illinois.v The resulting mixture wasthen slowly added to 974 grams of ground Rockland Mouse Dietandthoroughly mixed so that a final concentration of 1:1000 ofisonicotinic acid hydrazide to the diet was obtained. In an identicalmanner a diet containing a concentration of 111000 of isonicotinic acidhydrazide to a commercial diet known asBordens Dog Food was prepared.

The antituberculous activity of each of the diets. medicated withisonicotinic acid hydrazide as described above was determined in thefollowing manner: Groups of 20 mice each were in fected intravenouslywith 0.5 cc. of a dilution of a human strain of tuberculosis known as M.tuberculosis H37Rv in Dubos medium while other groups of 20 mice eachwere infected intranasally with 4 drops of a 10* dilution of M.tuberculosis H37Rv in Dubos medium. The Dubos medium employed is thatdescribed by Dubos, R. J. and G. Middlebrook: Media for TubercleBacilli, Am. Rev. Tub. 56: 834 (1947) containing the Tween but modifiedby the omission of the albumin. Ten animals from each of the groups wereallowed to feed ad libitum on the medicated diets for 21 days. Tenanimals from each group were placed on the plain unmedicated RocklandMouse Diet and on the Bordens Dog Food respectively, as controls. Thetreated animals consumed in this manner approximately 250 milligrams ofisonicotinic acid hydrazide per kilogram of body weight per day whengiven the 111000 diet. This figure is based on the observation that micenormally consume 5.0 grams of food per day, so that a 1:1000 dietcorresponds to a drug consumption of 250 mg./kg./day. The expressionmg./kg./ day means milligrams per kilogram of body weight per day. 7 e aAll of the animals were sacrificed and autopsied on the 22nd day. All ofthe animals which were fed on the diets containing isonicotinic acidhydrazide showed no gross tuberculous lesions in the lungs. On the otherhand, all control animals fed on the non-medicated diets showed Example2 250 mg. of isonicotinic acid hydrazide were dissolved in 250 cc. ofsterile distilled water, and another-250 mg. of isonicotinic acidhydrazide were dissolved in 250 cc. of 0.85 per cent sterile salinesolution, to give solutions of 1:1000. To test each solution of theisonicotinic acid hydrazide, groups of 20 mice each were infectedintravenously with M tuberculosis H37Rv while other groups of 20 miceeach were infected intranasally as described in Example 1. afterinfection, 10 mice from each group were subcutaneously injected. withthe isonicotinic acid hydrazide solutions, the other 10 mice serving ascontrols. The dose was given according to the weight of the mice,calculated on the basis of 1 cc. per 20 grams body weight, so that eachanimal received 50 mg./kg./day of isonicotinic acid hydrazide. Atotal of21 daily subcutaneous inj ections were given to the animals.

The animals were sacrificed and autopsied on the 22nd day. All treatedanimals showed no spread gross tuberculous lesions.

'In other experiments carried'out in the same manner, but with differentconcentrations of isonicotinic acid hydrazide, the lower limit of ac forthe intravenous and intranasal infection,

"5.0 mg./kg./day or one gram of isonicotinic acid Shortly grosstuberculous lesions in the lungs. All con-Q trol animals, on the otherhand, sh'owed widehydrazide in 10,000 cc. of sterile distilled water or0.85 per cent saline solution.

Example 3 body weight so that each animal received 50 mg./kg./day ofisonicotinic acid hydrazide. A total of 21 daily intra-abdominalinjections were given to all treated animals. I

The animals were sacrificed and autopsied on the 22nd day. All treatedanimals showed no gross tuberculous lesions in the lungs. All controlanimals on the other hand, showed widespread tuberculous lesions. Inother experiments carried out in the same manner, but employingsolutions of different concentrations of isonicotinic acid hydrazide,the lower limit of activity of the solutions, that is, th smallest doseof isonicotinic acid hydrazide giving 100 per cent protection of theanimals, was found to be 1.25 mg./kg./day; that is, 1 gram ofisonicotinic acid hydrazide in 40,000 cc. of sterile distilled water, or0.85 per cent sterile saline solution, for the intravenous infection;and 2.5 mg./kg./day, thatlis, 1 gram of isonicotinic acid hydrazide in20,000 cc. of sterile distilled water, or sterile 0.85 per cent salinefor the intranasal infection.

Example 4 1 250, mg. of isonicotinic acid hydrazide were dissolved in250 cc. of sterile distilled water to give a solution of 1:1000. Groupsof 201mice each were infected intravenously and other groups of 20 micewere infected intranasally in the same manner as described in Example 1.Shortly after the infection, the solution was administered per os to 10mice from each group by stomach tube, the other 10 mice serving ascontrols. The dose was administered according to the weight of the micecalculated on the basis of 1 cc. per 20 grams body weight, so that eachanimal received 50 mg./kg./day of isonicotinic acid hydrazide. A totalof 21 daily per os treatments were administered to the animals bystomach tube. The animals were sacrificed and autopsied on the 22nd day.All treated animals showed no gross tuberculous lesions in the lungs.All control animals, on the other hand, showed wide-spread tuberculousgross lesions.

In other experiments carried out in the same manner, but employingsolutions of different concentrations of isonicotinic acid hydrazide,the lower limit of activity of the solutions, that is, the smallest doseof isonicotinic acid hydrazide giving180-l00 per cent protection of theanimals, was found to be 6.3 mg./kg./day; that is, 1 gram ofisonicotinic acid hydrazide in about 8,000 cc. of sterile distilledwater for the intravenous infection, and 2.5 mg./kg./day, that is, 1gram in 20,000 cc. of sterile distilled water for the intra- I nasalinfection. I

The efilcacy of isonicotinic acid hydrazide in vitro is shown in thefollowing example.

Example 5 9 cc. of Dobos medium were placed into the first test tube ineach of a series of eleven test tubes. Into the remaining test tubeswere pipetted 5 cc. of the same medium. One cc. of a 1:200 solution indistilled water of isonicotinic acid bydrazide was placed into the firsttube in each series and mixed thoroughly with the 9 cc. of the Dubosmedium thus giving a final concentration of 1:2000 isonicotinic acidhydrazide. Serial dilutions were then made through the next 9 tubes ineach series by transferring 5 cc. from the first tube tothe second, etc.The 10th tube in each series thus contained a concentration of111,024,000 of isonicotinic acid hydrazide.v Thev 11th tube in eachseries served asa control. Each test tube was then inoculated with onedrop ora 7-10 day old culture of M. tuberculosis H37Rv and incubated at37 C. Readings were taken at 7, 14 and 21-day intervals. No growth of M.tuberculosis occurred in any of the tubes containing isonicotinic acidhydrazide. The controls, however. developed normalgrowth of theorganism.

Additionalinvestigations have been carried out which confirmandestablish the results shown in,

Example 5 with respect to the activity of isonicotinic acid hydrazide aswell as acid addition salts thereof. The in vitro tests supplement thetests,

used with various animals, rodents and tests on human beings. All ofthese various investigations have cumulative significance with respectto my,

invention.

Treatments of monkeys has also been carried out. Note the report ofIrving Zieper and Roger Lewis, Tuberculosis in a Macacus Rhesus Treatedwith Isonicotinylhydrazine, The Quarterly Bulletin of Sea View Hospital,vol. XIII, No. 1, January 1952, pages 12 to 16. Therein the conclusionappears that isonicotinylhydrazine was efiective for combatting}clinical tuberculosis of monkeys.

Of prime interest is the value of the hydrazide compositions forcombatting tuberculosis in humans. Reports by Irving Selikofi, EdwardRobitzek and George Ornstein, The Quarterly Bulletin, upra, pages 17 and27, present data and conclusions as to chemotherapy and toxicity..

The authors state that the absence ofserious toxic effects on liver,kidney and bone marrow is in agreement with primate experimentsconducted by Lewis and Zieper. Robitzek, Selikoff and Ornstein,Quarterly icity, return of temperature to normal, recovery of appetite,remarkable weight gain, all with a rapidity and certainty to a degreenever observed by them in other chemotherapeutic or antibiotic agents.They also note important chemotherapeutic efiects on extra-pulmonarytuberculosis, including meningeal and oro-pharnygeal and laryngealtypes, to a degree superior to experiences with known antibiotic orchemotherapeu--- tic agents.

As noted above, my compositions for comb atting tuberculosis may takeany of a variety oi. forms. Various diluents maybe employed. Ofparticular interest is the use of animal feed compositions, such as themouse feed and dot teed Bulletin, supra, pages 27-51, report on theohemo-' compositions containing the active ingredient, as"

outlined above. Other animal feeds may be used consistent with theparticular animals to whom the compositions are administered.

The percentage of active ingredient in my compositions may be varied. Itis necessary that the active ingredient constitute aproportion-suchth'at a-suitable dosage will-be Obtained. Obviously=several unit dosage forms may be administered at "about the same time."Although I have found that a percentage if-"less than 0.10 per cent ofactive ingredient iseffective'l prefer to use not less than 0.10 percent of active agent.- Activity increases-withconcentration of theagent. I have found that'- the percentage of active agent maybelo-percent. or 2'5 -p'er cent, or even a higher proportionFor""exafrlple tablets may be prepared with a minor proportion ofdiluent and a major proportionof -active material. 'Iablets containingfrom about l'to'about 50 mg. of active ingredi-' ent are-particularlyuseful. The following formul ations, as-well as those already givenabove, are intended to be illustrative only and they may be varied ormodified .to a considerable extent without departing from the spirit ofthe invention. I do not therefore intend to limit'myinvention to thespecific embodiments herein set forth.

. Sugar coating, approximately 50 .IThe powders 1, 2, and 3 are mixed,then. granulated with 4. Add 5 and 6 and tablet... Coat withsugarcoating.

Example 8 Mg. 1. Isonicotinic acid hydrazide 10 2. Cornstarch 1 -Q 5'73. Lactoselulne 4. T"alc. r a 9 5...Stearic acid 6 f QPowde'rs'L 2, andwere'slugged, then granulated, mixed with 4 and 5;'and tableted. 1

Isonlcotinic acid'hydrazide"; s e mg; 25 Placebo granule" mg 160 1 Ing'Stearic acid ..mg,-. 5 Magnesium stearate mg 1:. The placebo granulescontained:

Lactose per cents- 55 Procedure.-'- A placebo granule is madeoi lactoseand %'cornstarch. Isonicotinic acid hydrazide, screened through #20mesh; is added;

and the lubricants, talc, stearic acid and magnesium stearate, areadded. Tableting a rotary machine. I

Example 11 An illustrative example of preparinga largelo't of ampulsolution is as follows:

Eight liters of distilled water for injection} U. S. P., are placed in a20-liter Pyrex'glass bottle. To this water 500 grams of isonicotinicacid hydrazide are added with stirring until solution is effected. ThepH i adjusted thereafter with sufiicient reagent grade N/lO hydrochloricacid to achieve a pH of about 6.0. Then sufficient water for injectionisadded to bring the volume to 10 liters. The solution is filtered andfilled into clean, dry, sterile, flint glass ampuls, ea'ch containing2.2 cc. of the fluid. The ampuls are sealed and sterilized for 30minutes at 115 F.

Iclaim:

a pH of about 6.0.

2. A composition for combatting tuberculosis comprising not less than0.10 per cent of a mem-' ber selected from the group consisting ofiso-fl nicotinic acid hydrazide and acid" addition sans".

thereof and asterile parenteral water diluent adjusted to a pH of about6.0 withv hydrochloric acid.

3. A veterinary feed for co'mbatting tuber:

culosis comprising not less than OJO'per cent of a member selected fromthe group'consist.

ing of isonicotinic acid hydrazide and acid addition salts thereof.

4. A composition in dosage unit form for co batting tuberculosiscomprising not less than10' mg. of a member selected from thegroupconsisting of isonicotinic acid hydrazide and acid adsolid ditionsalts thereof per dosage unit and a pharmaceutical carrier.

5. A composition in dosageunit batting tuberculosis comprising about 10to about mg. of isonicotinic acid hydrazide per dosage unit and a solidpharmaceutical carrier;

6. A 'compo'sition in dosage tablet unit form for combattingtuberculosis comprising about 10 to about 50 mg. of isonicotinic acid'hydrazide per dosage unit and a. solid pharmaceutical carrier. W7. Acomposition in dosage unit formfo'r combatting tuberculosis comprisingnot less 1 than" about 10 mg. of a member selected from the groupconsisting of isonicotinicacid hydrazide and acid addition salts thereofanda solid pharmaceutical carrier, said composition being in tabletdosage unit form.

8. A composition according to claim 7 in which the-diluent comprises atleast one member se-' is done on,

1. A composition for combatting tuberculo is, comprising not less than0.10 per cent of a mem ber selected from the group consisting of isO-Inicotinic acid hydrazide and acid addition saltsthereof and a sterileparenteral water. diluent at.

forin for com- 1 accaoco lected from the group consisting of lactose,cornstarch, and stearic acid.

9. A composition for combatting tuberculosis comprising from about 0.1per cent to about 50 per cent of a member selected from the groupconsisting of isonicotinic acid hydrazide and acid addition saltsthereof and a significant amount of a pharmaceutical carrier.

10. A composition for combatting tuberculosis comprising from about 0.1per cent to about 50 per cent of isonicotinic acid hydrazide andsignificant amount of a pharmaceutical carrier.

11. A composition for combatting tuberculosis comprising from about 0.1per cent to about 50 per cent of a member selected from the groupconsisting of isonicotinic acid hydrazide and acid addition saltsthereof and a significant amount of a solid phamaceutical carrier.

12. A composition for combatting tuberculosis comprising from about 0.1per cent to about 50 per cent of isonicotinic acid hydrazide and asignificant amount of a solid pharmaceutical carrier.

13. A composition in dosage unit form for combatting tuberculosiscomprising from about 1 mg. to about 50 mg. of a member selected fromthe group consisting of isonicotinic acid hydrazide 10 and acid additionsalts thereof and a significant amount of a pharmaceutical carrier.

14. A composition for combatting tuberculosis comprising not less than0.1 per cent of a member selected from the group consisting ofisonicotinic acid hydrazide and acid addition salts thereof and acarbohydrate containing pharmaceutical carrier.

. 15. A composition in dosage unit form for combatting tuberculosiscomprising not less than 10 milligrams of isonicotinic acid hydrazideper dosage unit and a solid pharmaceutical carrier.

HERMAN HERBERT REFERENCES CITED The following references are of recordin the tile of this patent:

9. A COMPOSITION FOR COMBATTING TUBERCULOSIS COMPRISING FROM ABOUT 0.1PER CENT TO ABOUT 50 PER CENT OF A MEMBER SELECTED FROM THE GROUPCONSISTING OF ISONICOTINIC ACID HYDRAZIDE AND ACID ADDITION SALTSTHEREOF AND A SIGNIFICANT AMOUNT OF A PHARMACEUTICAL CARRIER.